PROJECT SUMMARY/ABSTRACT White and brown adipose tissues are highly vascularized organs, capable of plasticity based on metabolic demands and energy expenditure. Critical gaps remain in our understanding of how angiogenesis impacts adipose tissue dysfunction and overall metabolism. MicroRNAs (miRs) are implicated in the regulation of the angiogenic response to pathophysiological stimuli. However, the role of miRs in regulating the angiogenic response in diet-induced insulin resistance is poorly understood. Using a miRNA-Seq approach, we identified that miR-409-3p expression was significantly increased in endothelial cells (ECs) of brown adipose tissue (BAT) of diet-induced obese (DIO) mice and in human diabetic plasma samples compared to non-diabetic patients. Overexpression of miR-409-3p markedly inhibited EC growth and migration, whereas miR-409-3p inhibition had the opposite effects. Preliminary studies indicate that miR-409-3p targets the 3'UTRs of Zinc Finger E-box binding Homeobox 1 (ZEB1) and Mitogen-activated protein kinase kinase kinase kinase 3 (MAP4K3). SiRNA knockdown of ZEB1 or MAP4K3 expression in ECs phenocopied the effects of miR-409-3p overexpression and significantly decreased EC proliferation and migration. 3T3-L1 cells or human skin fat organoids co-cultured with conditioned media from ECs overexpressing miR-409-3p had decreased expression of BAT markers (UCP1, Cidea), whereas conditioned media from ECs deficient in miR-409-3p markedly increased expression of BAT gene expression. Mechanistically, Placental Growth Factor (PLGF), secreted from ECs in response to miR-409-3p inhibition promoted 3T3-L1 differentiation towards brown-like adipocytes as measured by increased UCP1 expression. Systemic delivery of LNA-anti-miR-409-3p inhibitor to DIO mice significantly increased angiogenesis as measured by CD31 staining, UCP-1 expression in BAT and subcutaneous white adipose tissue (sWAT), while improving glucose and insulin tolerance and energy expenditure. Therefore, we hypothesize that miR-409-3p serves as a critical regulator of EC growth and angiogenesis in adipose tissue and may improve metabolic dysfunction in DIO. Thus, in Aim1 we will investigate the molecular mechanisms by which miR-409-3p regulates EC growth and angiogenesis. In Aim2, we will delineate the mechanisms by which miR-409-3p in ECs regulates browning in adipose tissues. Finally, in Aim3, we will explore the effect of miR-409-3p neutralization in the vasculature of adipose tissues and development of DIO and insulin resistance in mice. Successful completion of these studies will shed insights on the regulatory role of miR-409-3p between impaired angiogenesis in diet-induced obesity and adipose tissue dysfunction, an effect that could be exploited for therapeutic intervention in obesity-induced insulin resistance. !